Ampure xp system

Total RNA of albino and green tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA integrity was assessed using the RNA 6000 Nano Assay Kit in the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. At last, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.

Total RNA of each sample was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA purity, concentration and integrity were checked, respectively. The library preparation and sequencing include the following steps. Firstly, ribosomal RNA was removed by Epicentre Ribo-zero™ rRNA Removal Kit (Epicentre, USA), and rRNA free residue was cleaned up by ethanol precipitation. Secondly, sequencing libraries were generated by NEBNext^® Ultra™ Directional RNA Library Prep Kit for Illumina^® (NEB, USA) using the rRNA-depleted RNA. Thirdly, double-stranded cDNA was synthesized replacing dTTPs with dUTPs in the reaction buffer used in second strand cDNA synthesis after RNA fragmenting. Fourthly, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments of preferentially 150∼200 bp in length. Fifthly, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer, and products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Finally, the clustering of the index-coded samples was performed on a cBot Cluster Generation System and the libraries were sequenced on an Illumina Hiseq 2000.